Tissue‐specific differences in Ca2+ sensitivity of the mitochondrial permeability transition pore (PTP). Experiments in male rat liver and heart

Abstract Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue‐specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.

] cyt is slightly under 100 nM, upon stimulation it may rise more than 10 times, to 1 μM in liver (Gunter et al., 1998) or 1.7 μM in the heart (Lu et al., 2016).The mitochondrial Ca 2+ uniporter (MCU) transports Ca 2+ into the mitochondrial matrix (Kovacs-Bogdan et al., 2014;Vais et al., 2020), while a slower Ca 2+ return to the cytoplasm is mediated by antiport with either Na + (mostly in heart) or H + (mostly in liver) (Belosludtsev et al., 2019).In fact, there are Ca 2+ hotspots in regions where the endoplasmic reticulum/mitochondrion come near, thus explaining the large amount of Ca 2+ needed to elicit half-activity by the Ca 2+ uniporter.In the extracellular compartment Ca 2+ is 10 4 times more concentrated than in the cytoplasm and its uptake makes it an ideal second messenger (Campbell, 2014).In addition, in different tissues the rate of mitochondrial Ca 2+ uptake is different, for example, Ca 2+ uptake in heart is faster than in liver due to low expression of the Ca 2+ uniporter control subunits MICU1 and MICU2 as compared to the channel subunit MUC resulting in a low MICU1/MUC ratio (Paillard et al., 2017).Thus, increasing MICU1 expression changes Ca 2+ uptake kinetics in the heart to make it comparable to the liver (Paillard et al., 2017).
From the above, it may be proposed that together with the detoxifying role of PTP opening, its efficient closure is needed for survival.Indeed, flickering has been reported in heart and muscle mitochondria (Boyman et al., 2019;Korge et al., 2011;Lu et al., 2016).Still, it is not known if PTP dynamics differ from tissue to tissue, that is whether in tissues that seldom see variations in [Ca 2+ ] Mit , such as the liver (Bagur & Hajnóczky, 2017;Burgess et al., 1984;Carafoli, 1987), PTP is as resilient as in tissues that are subjected to large, frequent Ca 2+ fluxes such as the heart (Abelmann, 1961;Akopova, 2001;Chalmers & Nicholls, 2003;Zhong et al., 2008).Here, PTP reversibility in isolated murine mitochondria from either heart or liver was compared to determine whether PTP properties vary from tissue to tissue.Heart mitochondria (Heart Mit ) PT remained reversible for longer periods and under higher Ca 2+ concentrations than liver mitochondria (Liver Mit ).Our results may have profound implications concerning the physiological role of the mitochondrial PTP in different organs.

| Animals
Wistar male rats weighing 200 g were fed with Laboratory Rodent Diet 5001 (LabDiet, Minnesota, USA), were obtained from our animal facilities at either the Instituto de Fisiología Celular (IFC, UNAM) or the Instituto Nacional de Cardiología (INC) and were housed at 22 ± 2°C on a 12 h/12 h light/dark cycle, with free access to a standard laboratory rodent chow and water.Before the experiment, each rat was fasted for 12 h and euthanized by decapitation.Experimental procedures using animals at the Instituto Nacional de Cardiología were authorized by the "Committee for the Care and Use of Laboratory Animals (C.I.C.U.A.L.)" with protocol number INC/CICUAL/008/2020.At IFC, UNAM, a local committee approved all experimental procedures according to Ethics in Animal Experimentation (SUV124-19).

| Isolation of mitochondria
Liver and heart were extracted from the same individual and placed in cold isolation buffer (250 mM sucrose, 10 mM Tris-HCl and 1 mM EDTA, pH 7.3).Both tissues were processed at the same time, but with different protocols.The differences were to the need to properly homogenize the highly resistant cardiac muscle (see below).In both tissues adequate respiratory controls were obtained, ensuring that membrane integrity was preserved (Corcelli et al., 2010;Pallotti & Lenaz, 2007).The heart was minced and incubated for 10 min in isolation buffer plus 1 mg proteinase K/mL, then it was centrifuged at 12063× g for 10 min, the pellet was washed with the same buffer before being homogenized with a Tissuemizer (model TR-10, Tekmar, Breisgau, Germany) at 3000 rpm × 10 s three times alternating with 1 min rest on ice.The homogenate was centrifuged a 1477× g for 5 min and the supernatant was centrifuged at 12063× g for 10 min.The mitochondrial pellet was resuspended and incubated with BSA 0.2% × 10 min and finally centrifuged 12063× g for 10 min and resuspended in reaction buffer (250 mM sucrose, 10 mM HEPES, pH 7.4) (Correa et al., 2007).At the same time, the liver was minced in isolation buffer as the heart, homogenized using a Potter-Elvehjem teflon homogenizer and centrifuged 1477× g for 5 min.Then the supernatant was centrifuged at 12063× g for 10 min.The pellet was suspended in reaction buffer (250 mM sucrose, 10 mM Tris-HCl and 20 μM EDTA, pH 7.4) (Gutiérrez-Aguilar & Baines, 2015).Mitochondrial suspensions from each source were kept in an ice-bath and used within 3 h.To ensure mitochondrial quality, respiratory controls were measured in an oximeter (see below) and mitochondria were used only when RC was at least 4.0 for liver or 6.0 for heart.

| Mitochondrial swelling
Swelling was followed as the decrease in absorbance at 540 nm (Jung et al., 1997).We used a DW2000 Olis/ Aminco spectrophotometer (GA, USA) in split mode.The concentrations of Ca 2+ and EGTA used are indicated under each figure.

| Oxygen consumption
Experiments were conducted using a high-resolution oxygraph (Oroboros, Innsbruck, Austria) at 30°C with continuous magnetic stirring.Reaction buffer for Liver Mit was 250 mM sucrose, 10 mM Tris-HCl and 20 μM EDTA, pH 7.4 (Gutiérrez-Aguilar & Baines, 2015) and for Heart Mit was 120 mM KCl and 10 mM HEPES pH 7.4 (Correa et al., 2007).In both experiments mitochondria (1 mg prot./mL) were added to a chamber containing 10 mM/10 mM glutamate/malate (G/M) and 5 mM phosphate (Pi).After measuring the basal O 2 consumption, one pulse of Ca 2+ was added and EGTA was added as indicated.

Ca 2+ retention capacity using either Arsenazo III or Ca 2+ Green-5N
In order to measure Ca 2+ fluxes in mitochondria, it was decided to use two alternative methods: the DW2C absorbance-based indicator arsenazo III or the fluorescent indicator Ca 2+ Green-5 N. For each method we used different reaction mixtures in an effort to eliminate any possible artifacts from our experiments.(a) Arsenazo III.Measurements of mitochondrial Ca 2+ retention capacity in mitochondrial suspensions (1 mg prot./mL) were performed at room temperature with continuous magnetic stirring in a DW2000 Olis/Aminco spectrophotometer in dual mode 675-685 nm (Petronilli et al., 1993;Scarpa & Azzi, 1968;Uribe & Devlin, 1994).The reaction mixture (150 mM sucrose, 50 mM KCl, 5 mM Tris, and 250 μM KH 2 PO 4 pH 7.4) was supplemented with 50 μM Arsenazo-III, 100 μM ADP, and NADHlinked respiratory substrates (10 mM malate, 10 mM glutamate).Ca 2+ was added in pulses 5 or 10 μM each 1.5 min (Petronilli et al., 1993;Scarpa & Azzi, 1968).(b) Calcium Green-5 N. Either Heart Mit or Liver Mit (1 mg/ mL) were incubated in the same reaction buffer plus glutamate/malate (5 mM/5 mM) and 100 nM Calcium Green-5N (Thermos Fisher Scientific, Walthman, MA, USA) for 1 min (Correa et al., 2007).Subsequently, CaCl 2 was added repeatedly as indicated until PT induction (Figure S3).Where indicated, either 10 μM CsA a mMPT inhibitor or 75 μM ruthenium red (RuR) a Ca 2+ uniport inhibitor were added.Fluorescence was measured in a PerkinElmer LS50B spectrofluorometer (MA, USA) using a magnetically stirred quartz cuvette.Temperature was 30°C.And the end of each experiment, saturated concentrations of Ca 2+ (3 M) and EGTA (400 μM) were used to titrate the sample to determine the maximun (F max ) and minimal (F min ) fluorescence respectively (not shown).Mitochondrial [Ca 2+ ] was calculated as described by Amigo et al. (2017): where F is experimental fluorescence.As suggested by the manufacturer, the dissociation constant used was Kd = 14 μM.

| Statistical analysis
One-Way Anova tests were performed.Data are presented as mean ± SD.Sample sizes are indicated in figure captions.Tests were considered significant at the 95% level of confidence (p < 0.05).GraphPad Prism version 8 (GraphPad Software, CA, USA) was used for statistical analysis and to draw graphs.

| RESULTS
The heart undergoes frequent depolarization events, while in contrast, other tissues such as the liver seldom see any activity.It was thus hypothesized that mitochondria in each of these tissues should exhibit different sensitivity to the wide Ca 2+ fluxes associated to cell depolarization.To test this, murine liver or heart mitochondria were isolated from the same individual.In each sample, mitochondrial PT and its reversibility were evaluated by measuring different activities.

| Oxygen consumption
In isolated mitochondria, Ca 2+ addition increases the O 2 consumption rate (OCR).When Ca 2+ is sequestered, the rate of O 2 consumption returns to its basal levels unless PTP is open and depletes ΔΨ (Figure 1A) (Batandier et al., 2004;Zhong et al., 2008).In Liver Mit , 30 μM Ca 2+ increased OCR and EGTA addition at 30 s reverted this increase.However, at 60 s or later EGTA was ineffective (Figure 1A Liver; Figure 1B, black columns).These results suggest that Liver Mit PT became irreversible at some point after 30 s.When the same experiment was conducted in Heart Mit , 30 μM Ca 2+ was also added and no effects on OCR were observed, EGTA had no further effects (Figure 1A Heart, two upper traces).Increasing added Ca 2+ to 150 μM did accelerate OCR, which returned to the basal rate independently of EGTA addition reverted suggesting that PTP was closed (Figure 1A heart, two lower traces; Figure 1B white columns).Thus, in Liver Mit PT was triggered by 30 μM Ca 2+ and became irreversible sometime between 30 and 60 s, while in the Heart Mit acceleration of O 2 consumption was observed only at 150 μM Ca 2+ and the rate of O 2 consumption returned spontaneously to basal levels even after 180 s.Furthermore EGTA had no further effects.The results suggest that after 30 s of Ca 2+ exposure, PTP became open in Liver Mit while it remained closed in Heart Mit (Figure 1).

| Ca 2+ -driven mitochondrial swelling
Ca 2+ is taken avidly by mitochondria (Carafoli, 1987;Halestrap & Davidson, 1990) triggering PT which in the presence of high KCl, PT evokes swelling (Jung et al., 1997).Thus, to evaluate PT, Ca 2+ was added to mitochondria in the presence of 20 mM KCl. Liver Mit did not swell spontaneously (Figure 2A, trace a).However, sequential 5 μM Ca 2+ additions led to rapid swelling beginning at the third addition (Figure 2A, trace b).In the presence of Cyclosporine A (CsA) swelling was inhibited partially (Figure 2A, trace c) further suggesting that swelling was due to PT.In control Heart Mit swelling was minimal (Figure 2B, trace a).Under these conditions repeated addition of 10 μM Ca 2+ induced mitochondrial swelling (Figure 2B, trace b).Again, CsA inhibited Heart Mit swelling (Figure 2B, trace c).Thus, the results in Figure 2 confirm that mitochondrial swelling was more evident and took place at lower Ca 2+ concentrations in liver than in Heart Mit .In addition when comparing to Figure 3B it was obvious that cardiac mitochondria did not undergo full swelling.

| Reversibility of Ca 2+ -driven mitochondrial PTP opening
In the presence of KCl, mitochondria exhibit only a mild rate of swelling (Figure 3, trace a); rapid mitochondrial swelling may be promoted by adding an adequate amount of Ca 2+ (Figure 3, trace b); CsA prevents the Ca 2+ effect, indicating that swelling is due to opening of PTP (Figure 3, trace c).In all cases, Ca 2+ quenching should stop swelling by closing PTP.This system was tested in Liver Mit and Heart Mit to explore PT reversibility (Figure 3).In Liver Mit full swelling was observed at 30 μM Ca 2+ while Heart Mit needed 150 μM Ca 2+ (Figure 3).In Liver Mit Cyclosporine-sensitive Ca 2+ -mediated mitochondrial swelling was triggered with 30 μM Ca 2+ (Figure 3A, trace b).Then, EGTA was added at different times, and it was observed that swelling stopped at 30 s (Figure 3A, trace d).When EGTA was added at 60 s (Figure 3A, trace e) swelling continued for 5 s and then stopped.At 120 s (Figure 3A, trace f) swelling was not inhibited.When the same experiment was conducted in Heart Mit , 150 μM Ca 2+ was needed to trigger similar swelling (Figure 3B, trace b).In addition, at all times evaluated, 30, 60 or 120 sec, EGTA stopped swelling immediately (Figure 3B, traces d, e and f, respectively).Results suggest that while hepatic mitochondrial PTP opening lost reversibility after 30 s, cardiac mitochondrial PTP remained reversible after addition at all three times tested.
F I G U R E 1 (A) Effect of Ca 2+ addition and quenching on OCR measured as nanoatom-gram of oxygen per minute per milligram of protein, (natgO min -1 mg protein -1 ).Liver Mit (left) or Heart Mit (right).Reaction mixture: 250 mM sucrose, 10 mM Tris, 5 mM Pi, 20 M EDTA, pH 7.4.Ca 2+ addition was 30 M to Liver Mit and either 30 or 150 M Ca 2+ to Heart Mit as indicated.EGTA was the same concentration as Ca 2+ .(B) Replot of data from triplicates of experiments in A. Liver Mit (black columns) and Heart Mit (white columns).Data are presented as mean ± SD of three independent experiments.Statistical differences from tissue to tissue are indicated in the figure with their p value.In addition, statistical differences within a tissue against its respective basal OCR are indicated as follows: Liver: latin letters; Heart, greek letters.For Liver Mit "a": p < 0.0001.For Heart Mit "α": p = 0.0243; "β": p = 0.0003, and "γ": p < 0.0001.

Arsenazo III
Many reports indicate that sequential Ca 2+ additions trigger mitochondrial PT (Haworth & Hunter, 1979;Scarpa et al., 1978;Uribe & Devlin, 1994).As expected from Figures 2 and 3, in Liver Mit the fourth to fifth 5 μM Ca 2+ addition triggered PT and the release for Ca 2+ (Figure 4A, trace a), while CsA delayed PT to about twice as many additions (Figure 4A, trace b).In contrast, in Heart Mit , repeatedly adding 5 μM Ca 2+ with or without CsA did not trigger PT (Figure 4B).Only when adding aliquots of 20 μM and  3.5 | Ca 2+ -retention assays using Ca 2+ -

Green
In order to confirm our data with Arsenazo III, Ca 2+ flows were measured with the fluorescent dye Ca 2+ Green-5N.In addition, a different reaction mixture with high KCl, and without previous treatment with chelex-100 was used (Correa et al., 2007) (Figure S3).Sequential additions of 20 μM Ca 2+ were tested: Liver Mit presented PT at the fifth addition (Figure S3A, black trace a) while Heart Mit resisted almost three times as many Ca 2+ additions before undergoing PT (Figure S1A, gray trace b).When a higher Ca 2+ (50 μM additions) was tested (Figure S3B), PT was exhibited at the second addition by Liver Mit (Figure S3B, black trace a) while Heart Mit underwent PT only the sixth to seventh addition (Figure S3B, gray trace b).Thus, the results were similar to those performed with Arsenazo III (Figure 4).Additionally, it was also confirmed that CsA inhibited PT in both cases (Figure S3C); except that in Figure S3C, an upward shift of Ca 2+ was observed in Heart Mit (Figure S3C, gray trace b) which was not present in the Arsenazo III experiment (Figure 4B).This was probably due to the addition of higher Ca 2+ used in the Ca 2+ -Green experiment and an active Ca 2+ antiport in the heart.Indeed, RuR inhibited Ca 2+ uptake (Figure S3D) by both Liver Mit (Figure S3D, black trace a) and Heart Mit (Figure S3D, gray trace b).
In addition to releasing ions from the matrix, PTP has been proposed to work as a physiological uncoupling mechanism that prevents mitochondrial ROS overproduction (Morales-García et al., 2021).Here, in Liver Mit PTP opening did not affect ROS concentration, but instead, after 2-4 min ROS increased slightly as compared to the control (Figure 5A).In contrast, in Heart Mit PTP opening slightly decreased ROS, although this was not statistically significant (Figure 5B).Further experiments are needed to determine whether in the heart PTP opening does decrease ROS production.A considerable decrease in ROS was expected (Kamunde et al., 2018).Further experiments subjecting mitochondria to different sources of stress have to be conducted to analyze this phenomenon.

| DISCUSSION
Mitochondrial structure, density and function may vary from tissue to tissue (Veltri et al., 1990;Heine & Hood, 2020).Physiological activities such as ROS generation, membrane potential, and mitochondrial redox status may also vary with the tissue and the metabolic status (Kuznetsov et al., 2004).Mitochondrial responses to substrates, inhibitors or stress may be different (Kristián et al., 2002).Brain mitochondria contain fewer and smaller mitochondria that kidney or liver, while the heart has the largest and most abundant mitochondria (Veltri et al., 1990).In heart and liver the mitochondrial reticulum organization is different (Aon et al., 2006;Kang et al., 2024).Even at the level of individual enzymes, the tissue-specific expression of subunit in Cytochrome c oxidase reflects different basal energy requirement in each organ (Vogt et al., 2021), and the Ca 2+ efflux (Serna et al., 2022).In regard to the mitochondrial calcium uniporter MUC, expression of the regulatory subunits MICU1 and MICU2 is higher in the liver than in the heart, which results in slower Ca2+ uptake kinetics by the liver (Paillard et al., 2017).
In human heart myocytes, Ca 2+ floods the cytoplasm 72 to 80 times per min and during intense cardiac activity heartbeats may increase to 160/min (Cheng & Lederer, 2008).Under these conditions, Ca 2+ saturates the mitochondrial matrix, so in an effort to empty matrix Ca 2+ , PTP opens and depolarizes the membrane.Then, PTP needs to be rapidly closed in order to avoid ATP depletion, OMM-disruption, Cytochrome c release and eventually, cell death (Kim et al., 2003;Morciano et al., 2021).In contrast, organs such as the liver are not subjected to these frequent depolarizations.Sometimes these tissues substitute Ca 2+ uptake as second messenger using cAMP-dependent signaling (Rodgers, 2022;Wahlang et al., 2018), so mitochondrial Ca 2+ overload seems to be less likely, that is Liver Mit face lower risk of Ca 2+ flooding and thus PTP does not need to be as dynamic.To evaluate this idea, it was decided to compare the robustness of the mitochondrial-PTP from liver versus that from heart.
Studies on whole tissues cells would complement our results (Agarwal et al., 2017;Boyman et al., 2019;Lu et al., 2016).In-situ mitochondrial studies can detect organelle heterogeneity within the cell or in a given tissue, for example in the liver, periportal mitochondria behave different to those in peripheral hepatocytes (Azimzadeh et al., 2016).Low-throughput analysis of small tissue/ cell culture samples has proven useful to study mitochondrial heterogeneity and dysfunction within a tissue (Bury et al., 2020;Liao et al., 2020).In isolated mitochondria, it is not possible to collect information on activity modulation by surrounding organelles or by the differences of irrigation and hormone concentrations in the cytoplasm (Hoek et al., 1997;King et al., 2024).Still, isolated mitochondria are needed to elucidate specific catalytic mechanisms in mitochondrial proteins (Horten et al., 2024;Koch et al., 2024).In addition, some substrates and inhibitors do not traverse the plasma membrane readily and thus tight control of in-situ mitochondrial activities is harder to achieve (Azimzadeh et al., 2016;Picard et al., 2011).There are advantages and drawbacks for studies on each in-situ and ex-vivo mitochondria.Most times, data are complementary.
When PTP behavior was compared in mitochondria isolated from either liver or heart, reversibility of PTP opening was lost earlier and with less Ca 2+ in the liver, as 30 μM Ca 2+ opened PTP, increasing the rate of oxygen consumption in state 4 (Figure 1A) and inducing mitochondrial swelling (Figures 2A and 3A).Heart Mit PTP remained reversible for much longer time (Figures 1B, 2B,  3B and 4B).
Reversibility of the permeability transition (PT) is vital as the drop in ΔΨ leads to depletion of ATP and outer mitochondrial membrane disruption (Kim et al., 2003), which in turn leads to cell death.In Liver Mit , PT reversibility was lost sometime after 30 s (Figure 3A) or three sequential additions of 5 μM Ca 2+ (Figure 4A).In contrast in Heart Mit PT remained reversible for at least 2 min (Figure 3B) and withstood as much as eight consecutive 20 μM Ca 2+ additions (Figure 4B,C).These experiments clearly show that PTP is much more resistant to Ca 2+ in the heart than in liver mitochondria.
Under the conditions tested, ROS concentrations did not change significantly.In heart mitochondria small decreased was observed during the first 3 min, which returned to control at 4 min.ROS variation might be enhanced if mitochondria are subjected stress and then PT is evoked (Bernardi et al., 2023;Brady et al., 2004;Odagiri et al., 2009).However, this will remain as a perspective of this study.
Mitochondrial PTP structure and physiology may vary widely in different organisms.The Debaryomyces hansenii PTP is closed by monovalent cations (Cabrera-Orefice et al., 2015).In Yarrowia lipolytica, it has been necessary to use ionophores and high Ca 2+ loads to force PT (Kovaleva et al., 2009).Also, in crustaceans such as Artemia franciscana, Litopenaeus vannamei, Lipidophtalmus louisianensis, Crangon crangon, and Palaemon serratus it has not been possible to induce PT by Ca 2+ overloading (Holman & Hand, 2009;Konrad et al., 2012;Menze et al., 2005;Rodriguez-Armenta et al., 2021).In regard to structure, elimination of individual proteins has not led to conclusive results (Azzolin et al., 2010;Hurst et al., 2017;Izzo et al., 2016;Zoratti & Szabò, 1995).Recently it has been proposed that at least the F 1 F o -ATPase and the adenine nucleotide transporter (ANT) form pores with differen sensitivities to inhibitors (Carrer et al., 2021).In fact both proteins or at least the ANT plus some subunits from the F 1 F o -ATPase need to be present in order to constitute a PTP (He et al., 2017;Neginskaya et al., 2022Neginskaya et al., , 2023)).Our results show that two different tissues from the same organism display different PTP behavior.This would not be exceptional, for example different uncoupling proteins (UCPs) with different physiological roles are expressed in different tissues (Schulz & Schlüter, 2023;Wu et al., 2019;Zhang et al., 2010) so it seems worth exploring whether there are tissue-specific differences in the structure/physiology of the mitochondrial ANT.In fact, in the heart, most ANT is isoform 1, while in the liver ANT isoform 2 is preponderant (Stepien et al., 1992).Even if these isoforms are quite similar in sequence, post-transcriptional differences have been reported.Thus, the isoform-specific properties of ANTs and their participation in mitochondrial PT would be worth exploring.
It is suggested that PTP function/regulation is tissue specific.In the heart, it may work as a physiological uncoupling system that detoxifies Ca 2+ and decreases ROS production (Boyman et al., 2019;Lu et al., 2016).This has been described in the yeast S. cerevisiae (Cabrera-Orefice et al., 2015;Guerrero-Castillo et al., 2012;Morales-García et al., 2021).The physiologic or structural basis for the difference in PTP behavior observed in each tissue is an interesting idea that complements data of widely different PTP regulation and function among species (Azzolin et al., 2010;Frigo et al., 2023).It would be interesting to explore if the frequent depolarization observed in cardiac myocytes favors association of proteins such as the Ca 2+ uniporter, the ANT, the F 1 F O -ATPase and possibly others while in other tissues in the same organism associations are different or non-existent.In this regard, at least the Ca 2+ antiport-driven efflux is different in heart where the counter-ion is Na + than in the liver where it is H + (Carafoli, 1987).

F
Mitochondrial swelling.Liver Mit (A) is more sensitive than Heart Mit (B).Reaction mixture as in Figure 1 plus 20 mM KCl. Swelling was induced with Ca 2+ additions as indicated.(a) Control; no Ca 2+ additions; (b) addition of Ca 2+ pulses every 1.5 min (5 μM in liver and 10 μM Ca 2+ in heart); (c) 10 μM CsA.Representative traces, n = 3 (for raw data see FigureS1).

F
I G U R E 3 Mitochondrial (M) PT reversibility in (A) Liver Mit and (B) Heart Mit .Reaction mixture: as in Figure1.Swelling was induced with 20 mM KCl and swelling control was evaluated (a).PTP opening was induced with 30 μM Ca 2+ in liver while in heart mitochondria 150 μM Ca 2+ were needed (b).Where indicated 10 μM CsA (c).EGTA: 30 μM for Liver Mit or 150 μM to heart Mit at 30 (d), 60 (e) and 120 (f) sec (upward arrows).Representative traces, n = 3 (for raw data see FigureS2).

F
Calcium retention assays in isolated mitochondria (M).Effect of Cyclosporine A. (A) Liver Mit , (B) and (C) Heart Mit .Reaction mixture: as in Figure 1 except 50 μM Arsenazo III.Opening of PTP was induced adding 5 μM Ca 2+ pulses in Liver Mit or, as indicated, 5, 20 and 30 μM Ca 2+ to Heart Mit every 1.5 min (a).In traces b, 10 μM CsA.Representative traces, n = 3.30 μM Ca 2+ did PTP open at 100 μM Ca 2+ (Figure4C, trace a).In addition, CsA delayed PT, even when 300 μM Ca 2+ was added (Figure4C, trace b).Again, our results indicate that PTP in Heart Mit withstands much higher Ca 2+ loading than Liver Mit and it remains reversible for much longer.
5 PT decreases ROS production in (B) Heart Mit but not in (A) Liver Mit .Reaction mixture: As in Figure 1.PTP opening o was induced with 30 μM Ca 2+ in Liver Mit or 150 μM in Heart Mit .PTP closing was induced with 30 μM EGTA in Liver Mit or 150 μM in Heart Mit at 30, 60, 90, 180, and 240 s after Ca 2+ was added.Data are mean ± SD n = 3, no statistical differences were observed.